Bone repair associated with fracture delayed union, bone nonunion and bone defect is one of the pending matters in the orthopedics field. For a long time, autogenous bone transplanting, allogeneic bone transplanting and related biomaterials have been applied for bone defect repair. However, for the autogenous bone transplantation, not only the bone source is greatly limited, but also at least 10% clinic complications may follow the bone operation. Furthermore, it requires longtime crawl replacing process after implantation. For the allogeneic bone implantation, there exists great immunity rejection and potential danger of disease dissemination. So, in the past few years, biomaterials and the biomedical products with special functions, therefore, have attracted close attention and have already been used in clinic. However, some reports indicate that the bioactivity and biodegradation of the traditional biomaterials cannot meet the clinic requirement and the therapeutic efficacies are not very good. These drawbacks greatly limit the wide application of biomaterials in clinic.
Bone Morphogenetic Protein (BMP), a kind of multi-functional morphogenesis factor with prominent biological activity in bone growth and repair, has provided new strategy for the therapy of bone nonunion and defects. As early as in 1965, Urist, an American doctor discovered that some substances could induce ectopic bone formation in the decalcifying bone matrix, and named them as bone morphogenetic protein or BMP. To date, over 20 BMP family members have been isolated and characterized, classified as BMP-1, BMP-2 . . . and so on. BMPs (excluding BMP-1) belong to transforming growth factor-beta (TGF-β) superfamily. BMPs not only exert the regulation of various organs growth and cell oriented differentiation in early embryonic tissue patterning phase, but also induce mesenchymal stem cells within organisms irreversibly to cartilage and osteoblast after procreation, thereby playing an important role in bone and tooth formation as well as wound healing. During the concrescence process, the expressing levels of BMPs in the pathological site significantly increase and the BMPs are confined to fracture callus domain. BMP implanted into the soft tissue can induce new ectopic bone, which has already been used as evidence to investigate the bioactivity of BMP. So, BMP possesses the potential of gigantic fundamental research value and wide clinical application.
Among all growth factors, BMP-2 is proven to be the most effective for the bone forming. Up to date, the structure and function of BMP-2 have been deeply addressed. The natural BMP-2, as a non-collagen acid glycoprotein, is hydrophobic, insoluble in water, and easy to dissolve in high concentration urea and guanidinium hydrochloride. Because of the insolubility, it is difficult to be extracted from natural resource or be purified when expressed as the recombinant protein. In addition, BMP-2 molecule has a hydrophobic core and 30% acidic amino acids, so the pI thereof is about 5.0. It is well-established that the three pairs of intrachain disulfide bond and one pair of interchain disulfide bond derived from the seven conserved cysteines residues in the primary structure are critical for maintaining the natural active conformation of BMP-2. If the disulfide bonds in the BMP-2 molecule are reduced, the bioactivity will disappear completely. The mature BMP-2 molecule is in the form of dimmer which is consisted of 2 monomers linked by disulfide bond. Each monomer is composed of 114 amino acids, contains glycosylation site and molecular weight thereof is about 13 KD.
Sampath et al. had analyzed the structure of an osteoinductive protein extracted from ox bone matrix directly and found that even after deglycosylation, the dimer composed of 16 KD and 14 KD polypeptides still has the bioactivity to induce bone forming, indicating that glycosylation is not essential for its activity. That is to say, it is possible to adopt prokaryotic expression system to prepare BMP-2.
In vivo, the precursor of BMP-2 is synthesized with larger molecular weight, composed of signal peptides and carboxyl terminus (C-terminus) including 100-125 amino acids. There are 7 conservative cysteine residues at the C-terminus of BMP-2 molecule, which play an important role in the formation of dimer. After the C-terminus is splitted and released, 2 monomers combine with each other via disulfide bond to form dimer and then the active syn-chain or iso-chain dimmers are secreted. BMP-2 precursor does not have any latent organism recognition sequence Arg-Gly-Asp, which exists in precursor sequence of TGF-β1 and TGF-β2. The N-terminus of the mature peptide is rich in basic amino acids, making BMP-2 precursor easily adhere on extracellular matrix and the biological half-life of the BMP-2 is prolonged. Consequently, the bioactivity of bone formation or signal gradient of hBMP-2 in stage of development and differentiation are enhanced. [Biochem Biophys Res Commun, 2004; 318(3): 704].
BMP-2 can be purified from animal tissue (p-BMP-2) or expressed as recombinant protein (rhBMP-2). As early as in 1979, Urist et al [UAnn Thorac Surg, 1990; 49 (6):864-5] first dissociated and purified BMP-2 from rabbit decalcified bone successfully, extracted the ox bones morphogenesis protein (bBMP) in 1982 from the ox bones, and in 1987, Urist [Clin Orthop Relat Res., 1987 (214):295-304] established a set of standard procedures to extract BMP from human and ox bones. At present, most of p-BMP-2s are obtained from the normal bones of animals, such as ox, pig, sheep, horse, rabbit, mouse and so on. Although BMP widely exists in various animal bone tissues, the content thereof is very tiny, only several micrograms BMP in 1 kilogram of wet weight fresh bone. And for the BMPs from different sources, there are major differences in physicochemical property and molecular structure, as well as in activity for inducing bone-formation and stability. Also, since various BMPs combine with the insoluble non-collagenous protein (iNCP) tightly, it is very difficult to obtain unitary BMP. Therefore, extraction of BMP from animal bone is impeded by its complicated process, poor reproducibility, low yield, and low protein purity. Further, the renaturing process is complicated and it is difficult to maintain the protein activity. At the same time, the protein extracted from animals, when applied in the human body, may cause immunological rejection and risk of spreading of pathogen. Therefore, the BMP-2 extracted from animal is difficult to satisfy the demand of experiments and clinical applications.
The method of producing human BMP-2 by recombinant cells, not only can ensure large-scale production, but also can avoid immunological rejection, which has attracted great attention. Since Wozney et al. obtained the BMP-2 gene from ox bones and BMP-2 was expressed successfully in recombinant Escherichia coli in 1988, this method has been used to produce mass-production of human BMP-2 (Wozney J M, Rosen V, Celeste A J, et al, Novel regulators of bone formation: molecular clones and activities, Science, 1988; 242(4885):1525-34).
At present, the expression systems used for BMP-2 expression include both eucaryotic and procaryotic ones. Wozney J M (Wozney J M, Overview of bone morphogenetic proteins, Spine. 2002, 15; 27 (16 Suppl 1): S2-8) presumed that the recombinant hBMP-2 in COS-1 cells had the ability to induce cartilage but not bone formation. Zhao Ming has expressed recombinant human BMP-2 with inducing bioactivity in the eucaryotic cell COS and CHO. In Genetics Institute, Wozney et al. cloned hBMP-2 from the cDNA library of human U-20s cell and found that the full-length cDNA of BMP-2 had 1587 bp, encoding a 396 aa polypeptide. And the mature peptide of 114 amino acids with activity under the action of protease was obtained [Sugiura T., Biochem J. 1999, 338 (Pt2): 433-40]. US Patent Application No. 118363 revealed that COS, CHO etc. could be used to express rhBMP-2.
In eukaryotic cells, most nascent polypeptides undergo one or more types of posttranslational modification, such as glycosylation and disulfide bond formation, which prepare each molecule for its functional role and/or for folding into its biologically active conformation. From this viewpoint, the eukaryotic cell is the ideal expression system to obtain the recombinant hBMP with high biological activity. In fact, the first approved rhBMP-2 protein with the bioactivity to induce the bone formation in vivo was expressed by eucaryotic cells. It was also found that the cell culture medium of the recombinant cell expressing the BMP-2 gene did not show any activity to induce bone formation. Only after purification, can it show a dosage-dependent bone-forming activity. At present, the Infuse™ as rhBMP-2 Bone Graft produced by Medtromic Sofamor Danek Company using eukaryotic cell as the expression system has already been used for vertebral column coalesce and bone defect repair. Also, the OP-1™ as rhBMP-7 produced in CHO cell by Stryker Biotech Company has already been approved by FDA and has been applied in clinic. Unfortunately, the shortcomings associated with eukaryotic expression system includes low yield, high production cost and so on. As a result, the bulk demand of scientific research and clinic is not satisfied.
Compared with the eukaryotic expression system, gene manipulation in the prokaryotic expression system is relatively easy and the gene expression level is high. Additionally, the prokaryotic cell has cell wall, low nutrition requirement, good toleration to culture circumstance, thus resulting in relatively low production cost and high yield. Although, BMP can not be glycosylated in prokaryotic expression system, and expression products appear mostly in the form of inclusion body. As previously stated, glycosylation is not essential for the biological activity of BMP-2 [J. M. Wozney, Science, 1988, 24 2:15 28-15]. Therefore, prokaryotic expression system is also suitable for the expression and production of BMP.
Kubler N P [Int J oral Maxillofac Surg, 1998, 27:30] and Ruppert R [Eur J Biochem, 1996; 237: 295-302] have successfully expressed integrated mature hBMP-2 in E. coli. In China, different mature peptide genes of hBMP-2 with various lengths have been also successfully acquired in E. coli, and the ensuing studies indicated that the obtained recombinant hBMP-2 has some level of ectopic bone-formed activity. However, the researches in the world can not go further to carry out repeatable annealing and purify human mature peptide BMP-2 expressed in recombinant E. coli, which impede the commercialization of this product.
Lin Song et al. [Acta Biochimica et Biophysica Sinica, 1996, 28(1):8] discovered that the closer the nascent hBMP-2 got to its mature peptide length, the better bone-forming activity it can induce. In CN Patent 01116754.8, recombinant E. coli is constructed for the preparation of the truncated rhBMP-2-108 with 108 amino acids encoded by a DNA fragment of 324 bp. Although the invention can be applied to produce rhBMP-2 in industry, short biological half-life, poor in vivo stability and relatively low biological activity in comparison with the full length hBMP-2, restrict its wide application, especially in case of bone fracture delayed union, bone nonunion, and bone defect.
Modification of the protein structure is frequently used to change the amino acid sequences of the natural proteins, construct truncated and long chain type mutants, with the aim of enhancing expression level, protein activity and stability, such as long-chain type IGF-1 (insulin-like growth factors-1) and so on. Previously, the inventors developed a long chain rhBMP-2 to overcome the difficulties of renaturation, separation, low protein activity and instability in vivo, which greatly limited the industrialization of the rhBMP-2. And we have filed the invention entitled “the preparation and application of long chain recombinant human bone morphogenetic protein-2” (CN 1951964). In addition, optimization of DNA sequence according to the codon bias of the host cell is another important strategy to enhance expression level.
In summary, although it has been reported that BMP-2 was produced in both prokaryotic and eukaryotic recombinant cells, the production technology is still not satisfactory. Therefore, there is a great need to develop a new strategy to efficiently produce rhBMP-2 in E. coli and a suitable process for industrialization.